Assay Kits for Glucose Metabolism Research

The metabolism of glucose is central to mammalian life. Dynamic changes in any of the steps involved in
processing glucose and its derivatives contribute to a wide range of diseases. Measuring the enzymes and
metabolites is pivotal to biological and medical research. Cayman offers an array of tools to make these
measurements, quickly, easily, and accurately.

Glucose metabolism is a primary source of energy and biomaterials for the maintenance of cell homeostasis. Extra glucose is stored in the muscles and liver as glycogen which is hydrolyzed to glucose and released into the blood when needed. The rate of glucose uptake in cells is dynamic and tightly regulated by hormones and/or growth factors including insulin. Cancer cells exhibit increased glucose uptake and metabolism by aerobic glycolysis in order to support a high rate of proliferation. Chemicals that block glucose uptake by cancer cells have been shown to have anti-cancer effects. Cayman’s Glucose Uptake Cell-based Assay Kit provides a convenient tool for studying modulators of cellular glucose uptake. The kit employs 2-NBDG, a fluorescently-labeled deoxyglucose analog, as a probe for the detection of glucose taken up by cultured cells. Apigenin, a flavonoid that has been reported to be an inhibitor of glucose transport, is included as a control.

Glucose, a monosaccharide (or simple sugar), is the most important carbohydrate in biology. Transported via the blood stream, it is the primary source of energy for the body’s cells. Glucose level is tightly regulated in the human body. Failure to maintain blood glucose in the normal range leads to conditions of persistently high (hyperglycemia) or low (hypoglycemia) blood sugar. Diabetes mellitus, characterized by persistent hyperglycemia, is the most prominent disease related to failure of blood sugar regulation. Cayman’s Glucose Colorimetric Assay Kit provides a simple, reproducible, and sensitive tool for assaying glucose in plasma, serum, and urine. The glucose assay uses the glucose oxidase-peroxide reaction for the determination of glucose concentrations. In this assay, glucose is oxidized to δ-gluconolactone with concomitant reduction of the flavin adenine dinucleotide (FAD)-dependent enzyme glucose oxidase. The reduced form of glucose oxidase is regenerated to its oxidized form by molecular oxygen to produce hydrogen peroxide. Finally, with horseradish peroxidase as a catalyst, hydrogen peroxide reacts with 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine to generate a pink dye with an optimal absorption at 514 nm.

Cayman’s Glycolysis Cell-Based Assay Kit provides a colorimetric method for detecting L-lactate, the end product of glycolysis, produced and secreted by cultured cells. In this assay, lactate dehydrogenase catalyzes the reaction between NAD+ and lactate, yielding pyruvate and NADH. The NADH directly reduces a tetrazolium salt (INT) to a colored formazan which absorbs between 490 and 520 nm. The quantity of formazan produced is proportional to the quantity of lactate in the culture medium, and is thus an indirect measurement of glycolysis. This assay can be adapted to high-throughput screening to examine glycolytic regulators in a cell-based system. In addition, the non-invasive sampling leaves the cells intact and thus allows multiplex testing for additional markers.

Cayman’s Oxygen Consumption/Glycolysis Dual Assay Kit is a multi-parameter approach to measure cellular oxygen consumption and glycolysis in living cells. This assay utilizes MitoXpress® - Xtra, a phosphorescent oxygen probe, to measure oxygen consumption rate, and also quantifies extracellular lactate as a readout for glycolysis. Antimycin A, an inhibitor of the mitochondrial electron transport chain, is included as a control. The kit can be used for efficient screening of compounds that modulate mitochondrial and glycolytic function in cultured cells.

β-Hydroxybutyrate (β-HB; 3-hydroxybutyric acid) is a “ketone body” which is produced in the liver, mainly from the oxidation of fatty acids, and is exported to peripheral tissues for use as an energy source. The term ‘ketone body’ refers to three molecules, acetoacetate, β-HB, and acetone. β-HB and acetoacetate transport energy from the liver to the other tissues and acetone is generated by spontaneous decarboxylation of acetoacetate.1 The presence of ketosis may be normal or pathologic. Normally ketosis can indicate that lipid metabolism has been activated and the pathway of lipid degradation is intact. Normal ketosis is prevalent in many circumstances such as during fasting, after prolonged exercise or after a high fat diet. Pathological causes of ketosis include multiple organ failure, diabetes, childhood hypoglycemia, corticosteroid or growth hormone deficiency, intoxication with alcohol or salicylates and several inborn errors of metabolism.2 In acutely ill patients, these ketone bodies can accumulate in the body to cause ketoacidosis, which leads to the potentially life threatening condition known as metabolic acidosis.3 The presence and degree of ketosis can be determined by measuring blood levels of β-HB. Ordinarily, β-HB accounts for approximately 75% of the ketone bodies in serum.4,5,6 Measurement of β-HB provides a reliable index of the level of ketoacidosis, including the detection of subclinical ketosis.7,8,9 In diabetics, β-HB measurements (and blood glucose) can be used for the assessment of the severity of diabetic coma and is essential for the exclusion of hyperosmolar non-ketotic diabetic coma. The measurement of β-HB is also used to monitor insulin requirements, based on existing hyperketonemia.10 β-HB has more recently been evaluated for use in neurodegenerative diseases and inhibition of adipocyte lipolysis.11,12,13,14,15 Cayman’s β-HB (Ketone Body) Assay Kit provides a simple, reproducible, and sensitive tool for measuring β-HB levels in plasma, serum, urine, cell lysates, or tissue homogenates. The method for β-HB determination is based upon the oxidation of D-3-Hydroxybutyrate to acetoacetate by the enzyme 3-hydroxybutyrate dehydrogenase.16 Concomitant with this oxidation, the cofactor NAD+ is reduced to NADH. In the presence of diaphorase, NADH reacts with the colorimetric detector WST-1 to produce a formazan dye with an absorbance maximum at 445-455 nm. The absorbance of the dye is directly proportional to the β-HB concentration.

β-Hydroxybutyrate (β-HB; 3-hydroxybutyric acid) is produced in the liver, mainly from the oxidation of fatty acids, and exported to peripheral tissues for use as an energy source. Measurement of β-HB can provide a reliable index of the level of normal ketosis as well as detect pathological ketoacidosis, including the assessment of the severity of diabetic coma and monitor insulin requirements in diabetic patients. Cayman’s β-HB (Ketone Body) Fluorometric Assay Kit provides a simple, reproducible, and sensitive tool for assaying β-HB from plasma, serum, urine, tissue homogenates, and cell culture samples. The method for β-HB determination is based upon the oxidation of D-3-Hydroxybutyrate to acetoacetate by the enzyme 3-hydroxybutyrate dehydrogenase. Concomitant with this oxidation, the cofactor NAD+ is reduced to NADH. NADH reacts with the fluorometric developer to yield a highly fluorescent product which can be analyzed with an excitation wavelength of 530-540 nm and an emission wavelength of 585-595 nm. The fluorescence is directly proportional to the β-HB concentration.

Ascorbate (L-Ascorbic acid or Vitamin C) is a six-carbon lactone that is synthesized from glucose in the liver of most mammalian species, but not by humans. Therefore, humans must obtain ascorbate in their diet in order to survive. In humans, ascorbate acts as an electron donor for eight different enzymes. It also serves as an antioxidant and may be beneficial for reducing the risk of developing chronic diseases such as cancer, cardiovascular disease, and cataracts. Cayman’s Ascorbate Assay provides a reproducible, sensitive fluorescence-based tool for quantifying ascorbate from plasma, serum, urine, and fruit juices.

Glucose-6-phosphate (G6P) plays a key role in the homeostatic regulation of blood glucose levels by participating in glycolysis, glycogen synthesis, gluconeogenesis, and can be metabolized to NADPH, enabling protection against oxidative damage. Disruption of G6P activity leads to glycogen storage disease type I or von Gierke’s disease, a group of inherited metabolic diseases characterized by severe hypoglycemia, growth retardation, and hepatomegaly, due to accumulation of glycogen and fat in the liver. Cayman’s Glucose-6-Phosphate Fluorometric Assay provides a fluorescence-based method for detecting G6P in tissue homogenates and cell culture samples. In the assay, G6PDH catalyzes the oxidation of G6P to 6-phospho-D-gluconate, along with the concomitant reduction of NADP+ to NADPH. NADPH reacts with the fluorometric detector to yield a highly fluorescent product which can be analyzed with an excitation wavelength of 530-540 nm and an emission wavelength of 585-595 nm.

Glucose-6-phosphate dehydrogenase (G6PDH) is a cytosolic enzyme that catalyzes the first step in the pentose phosphate pathway. This pathway includes converting glucose to ribose-5-phosphate, a precursor to RNA, DNA, ATP, CoA, NAD, and FAD. The pathway also generates NADPH. G6PDH deficiency, the most common enzyme deficiency worldwide, causes a spectrum of diseases including neonatal hyperbilirubinemia, acute hemolysis, and chronic hemolysis. G6PDH activity has been shown to be upregulated in rat and mouse models of obesity, hyperglycemia, and hyperinsulinemia. Cayman’s Glucose-6-Phosphate Dehydrogenase Assay provides a fluorescence-based method for detecting G6PDH activity in a variety of samples including erythrocyte lysates, tissue homogenates, and cell culture samples.

Cayman’s pH-Xtra™ Glycolysis Cell-Based Assay Kit is designed to measure the extracellular acidification rate (ECAR), which is proportional to the rate of lactate production.1 This kit combines a glucose-based respiration buffer and inhibitors along with the fluorescent pH probe pH-Xtra™ allowing for the accurate determination of glycolytic rate in a high-throughput manner.2

L(+)-Lactate is the major stereoisomer of lactate formed in human intermediary metabolism. The lactate to pyruvate ratio reflects the redox state of the cell and describes the balance between NAD+ and NADH, which is dependent on the interconversion of lactate and pyruvate via lactate dehydrogenase (LDH). Monitoring lactate levels is, therefore, a good way to evaluate the balance between tissue oxygen demand and utilization and is useful when studying cellular and animal physiology. Cayman’s L-Lactate Assay provides a fluorescence-based method for detecting L-lactate in biological samples such as serum, plasma, blood, urine, and saliva. It can also be utilized to determine intracellular and extracellular lactate concentrations in cell culture samples. In the assay, lactate dehydrogenase catalyzes the oxidation of lactate to pyruvate, along with the concomitant reduction of NAD+ to NADH. NADH reacts with the fluorescent substrate to yield a highly fluorescent product. The fluorescent product is analyzed with an excitation wavelength of 530-540 nm and an emission wavelength of 585-595 nm.

Pyruvate (pyruvic acid) is a key intermediate in cellular metabolic pathways and is derived primarily from glucose via glycolysis. Abnormal blood pyruvate levels are reported in a number of disorders including shock, liver disease, congestive heart failure, diabetes mellitus, thiamine deficiency, and metabolic disorders. Cayman’s Pyruvate Assay provides a fluorescence-based method for quantifying pyruvate in biological samples such as serum, plasma, blood, urine, and saliva. It can also be utilized to determine intracellular and extracellular pyruvate concentrations in cell culture samples.

Glycogen is a polysaccharide that is the principal storage form of glucose in animal and human cells. Glycogen is made primarily by the liver and muscles, but it can also be made by glycogenesis within the brain and stomach. Cayman’s Glycogen Assay provides a simple, reproducible, and sensitive tool for assaying glycogen from tissue. In this assay, glycogen is hydrolyzed by amyloglucosidase to form β-D-glucose, which is then specifically oxidized to D-glucono-δ-lactone by glucose oxidase forming hydrogen peroxide in the process. Hydrogen peroxide, in the presence of horseradish peroxidase, reacts with 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) in a 1:1 stoichiometry to generate the highly fluorescent product resorufin which is measure at an excitation wavelength of 530-540 nm and an emission wavelength of 585-595 nm.

The oxygen consumption rate (OCR) of cells is an important indicator of normal cellular function. It is used as a parameter to study mitochondrial function as well as a marker of factors triggering the switch from healthy oxidative phosphorylation to aerobic glycolysis in cancer cells. Oxygen consumption is traditionally measured by a cumbersome oxygen electrode, a specialized piece of equipment that typically yields low sample throughput. The phosphorescent oxygen probe, MitoXpress® Xtra, developed by Luxcel Biosciences offers a novel method for analyzing oxygen consumption in whole cells. Cayman’s cell-based Oxygen Consumption Rate Assay Kit (MitoXpress® Xtra HS Method) utilizes this newly developed phosphorescent oxygen probe to measure oxygen consumption rate in living cells. Antimycin A, an inhibitor of the mitochondrial electron transport chain, is included to be used as a positive control. Glucose oxidase is also included in the kit to be used as a reference for oxygen depletion. The kit is easy to use and can be easily adapted to high throughput screening for compounds which modulate oxygen consumption rate.

Cayman’s Oxygen Consumption/Mitochondrial Membrane Potential Dual Assay Kit is a multiplex assay designed to provide a comprehensive picture of mitochondrial performance in living cells. This assay simultaneously measures both oxygen consumption rate and mitochondrial membrane potential by utilizing MitoXpress® Xtra, a phosphorescent oxygen probe, and the cationic dye, JC-1. Antimycin A, an inhibitor of the mitochondrial electron transport chain, is included to be used as a positive control. Additionally, glucose oxidase is provided as a reference for oxygen depletion. This easy-to-use kit can be adapted for high throughput screening of compounds that modulate mitochondrial function.

Cayman's ATP Detection Assay Kit - Luminescence provides a simple and effective tool for measuring total ATP levels. This assay uses firefly luciferase to convert ATP and luciferin to oxyluciferin and light. The light emitted in this reaction is directly proportional to the concentration of ATP present. Using the ATP Detection Standard, quantitative measurement of ATP content can be achieved with a dynamic range of 12 fmol to 10 pmol of ATP.